title: Efficiency of puromycin-based technologies mediated by release factors and a ribosome recycling factor.
abstract: Two puromycin-based techniques, in vitro virus (IVV) and C-terminal labelling of proteins, were developed based on the observation that puromycin binds the C-terminus of a protein. Puromycin technology is a useful tool for the detection of proteins and analysis of protein-protein interactions (PPIs); however, problems arise due to the existence of stop codons in the native mRNAs. Release factors (RFs) that enter the A-site of the ribosome at stop codons compete with puromycin. To overcome this issue, we have used a highly controllable reconstituted cell-free system for puromycin-based techniques, and observed efficient IVV formation and C-terminal labelling using templates possessing a stop codon. The optimal conditions of IVV formation using templates possessing a stop codon was RF (-), while that of C-terminal labelling was RF (-) and the ribosome recycling factor (RRF) (+). Thus, we have overcome the experimental limitations of conventional IVV. In addition, we discovered that RRF significantly increases the efficiency of C-terminal protein labelling, but not IVV formation.
authors.affiliation: Division of Interactome Medical Sciences, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai Minato-ku, Tokyo 108-8639, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Ohashi
authors.forename: Masamichi
authors.initials: M
authors.lastname: Ishizaka
authors.forename: Naoya
authors.initials: N
authors.lastname: Hirai
authors.forename: Etsuko
authors.initials: E
authors.lastname: Miyamoto-Sato
mesh_terms: D014176:Protein Biosynthesis; D015202:Protein Engineering; D025941:Protein Interaction Mapping; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011691:Puromycin / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism
chemical_list: D011506:Proteins; D012333:RNA, Messenger; D012269:Ribosomal Proteins; C087267:ribosome releasing factor; D011691:Puromycin
keywords: C-terminal protein labelling; in vitro virus; puromycin; ribosome recycling factor/release factor